Method and composition for inhibiting or slowing blood coagulation

ABSTRACT

A method and composition for inhibiting or slowing blood coagulation includes lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority on prior U.S. ProvisionalApplication Ser. No. 60/386,562, filed Jun. 7, 2002, and which isincorporated herein in its entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work leading to the present invention was supported by one or moregrants from the U.S. Government, including National Heart, Lung, andBlood Institute Grant R01 HL57867. The U.S. Government therefore hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention is generally directed to inhibiting or slowingblood coagulation, and more particularly to using lactadherin or afragment thereof as an agent for inhibiting or slowing bloodcoagulation.

There are several anticoagulant drugs which are in widespread clinicaluse and many others in development, including clinical trials. However,none of these agents have the mechanism of blocking access of bloodproteins to procoagulant membrane surfaces. Inhibition of coagulation atthis stage is an earlier step than most anti-coagulants target and, itis specifically an anticoagulant step as opposed to a step which wouldinhibit platelet aggregation or adhesion.

Investigators have sought anticoagulants that would work via thismechanism for decades. Phospholipases from snake venoms can function asanticoagulants by competing for membrane binding sites and the functionof several phospholipases have been analyzed in detail. These proteinsare unsuitable for use in humans because the corresponding enzymaticactivity (phospholipases cleave phospholipids releasinglysophospholipids and free fatty acids) cause inflammation and tissueinjury. A single class of agents, annexins, have been shown to inhibitblood coagulation by blocking the membrane surface. These proteins havebeen evaluated as anticoagulants in animals. The efficacy is modest, atleast partly because annexin is more fastidious in its requirements formembrane lipids than most blood clotting proteins. Thus, much of theprocoagulant membrane of cells remains unblocked, even with a vastexcess of annexin V. We compared the anticoagulant efficacy of annexin Vdirectly to that of lactadherin.

Lactadherin is a MW 47,000 glycoprotein of milk fat globules. It hasalso been known as PAS-6/7, indicating the two glycosylation variants(Reference 1), bovine-associated mucoprotein, BA-46, P47, and MFG-E8(Reference 2). Lactadherin has a domain structure of EGF1-EGF2-C1-C2 inwhich EGF indicates epidermal growth factor homology domains, and the Cdomains share homology with the discoidin family including thelipid-binding “C” domains of blood coagulation factor VIII and factor V(FIG. 1) (Reference 2). The second EGF domain displays an Arg-Gly-Aspmotif (Reference 3) which binds to the α_(v)β₅ and α_(v)β₅ integrins(References 1 and 4-6). The second C domain binds to phospholipids(Reference 6).

In milk fat globules, lactadherin lines the surface of the phospholipidbilayer which surrounds the central triglyceride droplet, apparentlystabilizing the bilayer (Reference 7). Lactadherin decreases thesymptoms of rotavirus infection in infants, possibly by binding torotavirus particles via carbohydrate moieties (Reference 8). In tissuesections, lactadherin is found localized on the apical portion ofsecretory epithelium in the breast (Reference 7). Abundant expression bybreast carcinoma tissue makes lactadherin a potential target forantigen-guided radiation therapy (Reference 9). Lactadherin is alsofound on the apical surface of epithelia in the biliary tree, thepancreas, and sweat glands (Reference 7) and is synthesized by aorticmedial smooth muscle cells (Reference 10). Function in these tissuesremains unknown. Lactadherin has been identified as a zonapellucida-binding protein on the acrosomal cap of sperm (Reference 11).

Blood coagulation factor VIII and factor V bind to phospholipidmembranes via “C” domains which share homology with lactadherin(References 12-14). Remarkable features of membrane binding for theseproteins include high affinity (K_(D) approx. 2 nM) (Reference 15) andsufficient specificity so that no plasma proteins compete for membranebinding sites (Reference 16). Factor VIII binds via stereo-selectiveinteraction with the phospho-_(L)-serine motif of phosphatidylserine(PS) (Reference 17). Factor V also exhibits stereoselective interactionwith PS (Reference 18). Binding of factor VIII is enhanced by thepresence of phosphatidylethanolamine (PE) in the membrane (Reference19), by unsaturated phospholipid acyl chains (Reference 20), and bymembrane curvature (Reference 19). The crystal structures of the C2domains of factors VIII and V suggest that membrane binding is mediatedby two pairs of hydrophobic residues displayed at the tips of β-hairpinturns (References 21-22). Mutagenesis studies have confirmed the role ofthese residues in phospholipid binding (Reference 23). The homology ofthe lactadherin C domains with those of factors VIII and V suggests thatsimilar phospholipid binding properties may exist. Indeed, lactadherinhas been found to bind selectively to PS (Reference 24) and to utilizeprimarily the C2 domain in its lipid binding (Reference 6).

Annexin V, like factor VIII and factor V, exhibits high affinity,PS-dependent membrane binding (Reference 25). However, the quadruplicatemembrane-binding motifs of annexin V are not homologous with thediscoidin-like domains of lactadherin, factor VIII, and factor V(Reference 26). Corresponding to the difference in structure, themembrane binding mechanism is different. In addition, annexin V requiresCa⁺⁺ for membrane-binding and the binding is chiefly hydrophilic innature (Reference 27). Annexin V does have the capacity to compete for afraction of the phospholipid binding sites utilized by the factorVIIIa-factor IXa enzyme complex and the factor Xa-factor Va enzymecomplex of the coagulation cascade so that it functions in vitro as amembrane-blocking anticoagulant (Reference 28). The well-definedmembrane-binding and anti-coagulant properties of annexin V make studieswith annexin V suitable controls for studies with lactadherin.

OBJECTS AND SUMMARY OF THE INVENTION

The principal object of the present invention is to provide an agentwhich inhibits or slows blood coagulation. The agent includeslactadherin, a fragment of lactadherin, a functional equivalent oflactadherin, or a functional equivalent of a fragment of lactadherin.

An object of the present invention is to provide an agent which inhibitsor slows blood coagulation by competing for membrane binding sites offactor VII and/or factor V.

Another object of the present invention is to provide an agent whichinhibits or slows blood coagulation by inhibiting the prothrombinasecomplex and the factor VII a-tissue factor complex.

Yet another object of the present invention is to provide an agent whichinhibits or slows clotting of whole blood.

Still yet another object of the present invention is to provide an agentwhich inhibits or slows coagulation reactions on cell membranes.

An additional object of the present invention is to provide an agentwhich is an efficient inhibitor of the prothrombinase and the factorXase complex regardless of the degree of membrane curvature and/or thephosphatidylserine content.

Yet an additional object of the present invention is to provide an agentwhich is more effective than annexin V in inhibiting or slowing bloodcoagulation or clotting.

Lactadherin, a glycoprotein of the milk fat globule membrane, containstandem C-domains with homology to discoidin-type lectins and tomembrane-binding domains of blood-clotting factors V and VIII. Weinvestigated whether the structural homology confers the capacity tocompete for the membrane-binding sites of factor VIII and factor V andto function as an anticoagulant. Our results indicate that lactadherincompetes efficiently with factor VIII and factor V for binding sites onsynthetic phosphatidylserine-containing membranes with half-maximaldisplacement at lactadherin concentrations of 1-4 nM. Bindingcompetition correlated to functional inhibition of factor VIIIa-factorIXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherinwas an efficient inhibitor of the prothrombinase and the factor Xasecomplexes regardless of the degree of membrane curvature and thephosphatidylserine content. Lactadherin also inhibited the factorVila-tissue factor complex efficiently whereas annexin V was lesseffective. Since the inhibitory concentration of lactadherin wasproportional to the phospholipid concentration, and because lactadherinwas not an efficient inhibitor in the absence of phospholipid, the majorinhibitor effect of lactadherin relates to blocking phospholipid sitesrather than forming inhibitory protein-protein complexes. Lactadherinwas also found to be an effective inhibitor of a modified whole bloodprothrombin time assay in which clotting was initiated by dilute tissuefactor; 60 nM lactadherin prolonged the prothrombin time 150% vs. 20%for 60 nM annexin V. These results indicate that lactadherin functionsas a potent phospholipid-blocking anticoagulant.

At least one of the above-noted objects is met, in part, by the presentinvention, which in one aspect includes blocking or reducing access to aprocoagulant molecule by a coagulation molecule by subjecting aprocoagulant molecule to lactadherin, a fragment of lactadherin, afunctional equivalent of lactadherin, or a functional equivalent of afragment of lactadherin.

Another aspect of the invention includes blocking or reducing binding ofa ligand to cell membrane binding site by subjecting a cell membranebinding site to lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.

Another aspect of the invention includes inhibiting or slowing bloodcoagulation by subjecting a predetermined amount of blood to aneffective amount of lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.

Another aspect of the invention includes inhibiting or slowing bloodclotting by administering to a subject in need thereof an effectiveamount of lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.

Another aspect of the invention includes preventing or reducinginflammation by administering to a subject in need thereof an effectiveamount of lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.

Another aspect of the invention includes inhibiting or slowing bloodcoagulation by administering to a subject in need thereof an effectiveamount of lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.

Another aspect of the invention is to provide an anticoagulant reagentwhich includes lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin. A pharmaceutical composition may be prepared by utilizingthe anticoagulant reagent with a pharmaceutically acceptable carrier ordiluent.

Another aspect of the invention includes removing a phospholipid from abiological fluid by providing a biological fluid including, or suspectof including, a phospholipid, subjecting the biological fluid to asuitable amount of a binding agent (lactadherin, a fragment oflactadherin, a functional equivalent of lactadherin, or a functionalequivalent of a fragment of lactadherin) allowing binding between thephospholipid and the binding agent, and removing the binding agent withthe phospholipid bound thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, novel features and advantages of thepresent invention will become apparent from the following detaileddescription of the invention, as illustrated in the drawings, in which:

FIGS. 1A-C illustrate competition for factor VIII and factor V bindingsites by lactadherin or annexin V;

FIGS. 2A-F illustrate the relationship of vesicle curvature and PScontent to inhibition of the prothrombinase complex by lactadherin orannexin V;

FIGS. 3A-F illustrate the relationship of vesicle curvature and PScontent to inhibition of the factor Xase complex by lactadherin orannexin V;

FIGS. 4A-B illustrate inhibition of the factor Vlla-tissue factorcomplex by lactadherin or annexin V;

FIG. 5 illustrates competition for factor IXa binding sites bylactadherin;

FIGS. 6A-C illustrate the relationship of inhibition by lactadherin tophospholipid concentration; and

FIG. 7 illustrates inhibition of whole blood prothrombin time bylactadherin or annexin V.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatlactadherin, a glycoprotein of milk fat globules, functions as ananticoagulant by competing with factors VIII and V for membrane bindingsites. Our experiments below show that lactadherin functions as a potentphospholipid blocking anticoagulant.

Materials

Human factor X, human factor Xa, and human factor IXa were obtained fromEnzyme Research Laboratories (Southbend, Ind.), human factor V, humanfactor Va, and corn trypsin inhibitor were obtained from HeamatologicTechnologies Inc. (Burlington, Vt.). Recombinant human factor VII was agift from D. Pittman of Genetics Institute, Cambridge, Mass. Humanfactor VIIa, human prothrombin, and human α-thrombin were obtained fromEnzyme Research Laboratories (South Bend, Ind.). Both recombinant humantissue-factor and lipidated recombinant human tissue-factor wereobtained from American Diagnostca Inc. (Greenwich, Conn.). Lactadherinwas a gift from Drs. C. W. Heegaard and J. T. Rasmussen of theDepartment of Molecular and Structural Biology, University of Aarhus,Denmark. Annexin V was obtined from Sigma. Bovine brain PS, egg yolk PE,and phosphatidylcholine (PC) were obtained from Avanti Polar Lipids(Alabaster, Ala.). Chromogenic substrate S-2238 and S-2765 were obtainedfrom DiaPharma Group Inc (Westchester, Ohio).

Methods

Evaluation and Storage of Proteins

Pure bovine lactadherin was supplied at a concentration of 1 mg/ml inphosphate-buffered saline. SDS-PAGE with silver staining revealed onlytwo bands corresponding to the previously described lactadherin doubletwith approximate MWs of 47 kDa and 50 kDa. Lactadherin was stored at−80° C., aliquotted after thawing and each aliquot subjected to lessthan 3 cycles of flash-freezing and rapid thawing. The purity andhandling of other proteins were as previously described (Reference 29).

Preparation of Phospholipid Vesicles

Phospholipid vesicles were prepared by evaporating chloroform from thedesired phospholipids (PS %: PE %: PC %=4:20:76 and 15:20:65),resuspending in methylene chloride and re-evaporating twice under argon.Phospholipids were then suspended by gently swirling tris-bufferedsaline (50 mM Tris, 150 mM NaCl, pH 7.4) over the dried lipid suspensionuntil all lipid was suspended. Vesicles prepared in this way were usedas large multilamellar vesicles (LMV) (Reference 30). Some of theresuspended vesicles were then sonicated in a high intensity bathsonicator (Laboratory Supplies, Inc. Hicksville, N.Y.) (Reference 31)and some were made extruding these phospholipid suspensions twenty timesthrough two stacked polycarbonate membranes (Millipore, Bedford, Mass.)in a High Pressure Extrusion Device (Sciema Technical Services,Vancouver, BC, Canada) under argon as described previously (Reference32). Phospholipid concentration was determined by phosphorus assay(Reference 33). Vesicles were used fresh, or 1 ml aliquots werequick-frozen in liquid nitrogen, stored at −80° C., and thawed at 37° C.Storage at 4° C. before incubation with microspheres or blood clottingfactors did not exceed 24 hours.

Relipidation of TF

Recombinant human tissue factor was relipidated into phospholipidvesicles of the indicated composition using theoctyl-β-D-glucopyranoside method (Reference 34). The nominal molarratios of TF to phospholipid monomer was 1:7500.

Fluorescein-Glu-Gly-Arg Chloromethyl Ketone Labeling of Factor IXa

Factor IXa was labeled with fluorescein-Glu-Gly-Arg chloromethyl ketone(Haematologic Technologies, Burlington, Vt.) essentially as describedfor the Dansyl-Glu-Gly-Arg chloromethyl ketone (Reference 35). Freefluorescein-peptide was removed by ultrafiltration (Centricon 30,Millipore Corp., Bedford, Mass.). Labeling efficiency, as judged by theratio of absorbance at 280 nm to absorbance at 490 nm, divided byextinction coefficients for factor IXa and fluorescein, was 0.2fluorescein-peptide:factor IXa.

Flow Cytometry Binding Assay

Lipospheres were prepared as previously described (Reference 16). Glassmicrospheres of 1.6 μm nominal diameter (Duke Scientific, Palo Alto,Calif.) were cleaned, size-restricted, incubated with sonicated vesiclesof the indicated composition, washed three times in 0.15 M NaCl, 0.02 MTris-HCl, 0.1% defatted bovine albumin, 10 μM egg PC as sonicatedvesicles. Lipospheres were stored at 4° C. and used within 8 hours ofsynthesis. Recombinant human factor VIII and purified human factor Vwere labeled with fluorescein maleimide as described (Reference 36).Fluorescein-labeled factor VIII (4 nM) or fluorescein-labeled factor V(4nM) were incubated with lactadherin or annexin V for 15 min at roomtemperature, the mixture was added to lipospheres for an additional 10minutes, and membrane-bound fVIII or factor V was measured by flowcytometry. This procedure was performed on 25-μL aliquots of 100-μLsamples with an approximate liposphere concentration of 1×10⁶/mL using aBecton Dickinson FACSCalibur flow cytometer. Data acquisition wastriggered by forward light scatter with all photomultipliers in the logmode. Noise was reduced during analysis by eliminating events withforward and side scatter values different from those characteristic ofthe lipospheres. Mean log fluorescence was converted to linearfluorescence for values depicted in the figures. Only experiments inwhich the fluorescence histogram indicated a log normal distribution, asjudged by inspection, were analyzed quantitatively. Flow cytometryexperiments were performed in 0.14 M NaCl, 0.02 M Trizma-HCl, 1.5 or 5mM CaCl₂ as indicated in figure legends, and 0.1% bovine albumin, pH7.5.

Mathematical Model

Competition of lactadherin for phospholipid binding sites of factor VIIIwas compared to the following model:fVIIIB/fVIIIB(max)=[fVIII]/((KD*(1+[Lactadherin]/Ki))+[fVIII])

-   -   where fVIIIB is membrane-bound factor VIII, fVIIIB(max) is        maximum bound factor VIII when the concentration of factor VIII        is saturating, K_(D) is the dissociation constant of factor VIII        with phospholipid binding sites, K_(i) is the dissociation        constant of lactadherin with the binding sites recognized by        factor VIII. The model assumes that phospholipid binding sites        are limiting. For the curves depicted in FIGS. 1-3, all data was        normalized to the calculated value of fVIIIB/fVIIIB(max) at the        indicated concentrations of factor VIII without lactadherin. For        factor Xase complex activity fVIIIB/fvIIIB(max) was assumed to        be proportional to residual activity. Curve fitting was by eye,        while varying Ki, as the concentrations of factor VIII and        lactadherin were known and the KD value for factor VIII        (Reference 30) had been determined experimentally (see FIGS.        1A-C, where displayed results are from a single experiment        representative of at least two experiments for all conditions).        The same model, substituting fV for fVIII, was used for        comparison to competition binding experiments with factor V and        inhibition of the prothrombinase complex.        Factor Xase Assay

The activation of factor X by factor IXa in the presence of lactadherinor annexin V was measured with a two-step amidolytic substrate assay(Reference 29) with the following modifications. Factor IXa, 0.1 nM, wasincubated with the specified concentrations of factor X and variedconcentrations of lactadherin and annexin V in 150 mM NaCl, 50 mM Tris,pH 7.4, 1.5 mM CaCl₂ (for 4% PS, sonicated vesicle) or 5 mM CaCl₂ (forthe other vesicles), 1 nM factor VIII, 0.1 nM thrombin for 5 min. at 25°C.; final reaction volume, 40 μl. The reaction was then stopped by theaddition of EDTA to 7 mM final concentration. The amount of factor Xagenerated was determined immediately using the chromogenic substrateS-2765 (0.31 mg/mL) on a Molecular Devices ELISA plate reader in kineticmode. The results displayed in the figures are means of duplicates froma representative experiment. For studies without phospholipid, 40 nMfactor IXa was mixed with 40 nM factor VIII and 250 nM factor X in thereaction buffer. The reaction was started by addition of 2 nM thrombinand 1.5 mM Ca⁺⁺. The reaction was allowed to proceed for 30 min. priorto quenching with EDTA and reading, as above.

Factor VIIa-Tissue Factor Assay

Relipidated tissue factor, at the indicated concentration, was mixedwith factor×(100 nM) factor Vila (100 pM) and varied concentrations oflactadherin or annexin V. The reaction was started by addition of 1.5 mMCaCl₂ and allowed to proceed for 5 min. at 25° C. The reaction wasstopped with EDTA and the quantity of factor Xa formed was determined asdescribed above for the factor Xase assay.

Prothrombinase Assay

Cleavage of prothrombin to thrombin was measured in a two-stepamidolytic substrate assay analogous to that for factor X activation aspreviously described (Reference 37). Factor Va, 1 nM; factor Xa, 6.2 pM;and lactadherin or annexin V at specified concentrations were incubatedfor 5 min. at 25° C. in a solution containing 150 mM NaCl, 50 mM Tris,1.5 mM (for 4% PS, sonicated vesicles) or 5 mM (for the other PS contentand/or vesicle size) CaCl₂, and 0.05% wt/vol ovalbumin, pH 7.8, prior toaddition of 1 μM prothrombin. After 5 min at 25° C., the reaction wasstopped by the addition of EDTA to a final concentration of 7 mM.Thrombin formation was assessed in a kinetic microplate readerimmediately after addition of 0.1 mM chromogenic substrate S-2238. Theresults displayed in the figures are means of triplicates from arepresentative experiment.

Activated Partial Thromboplastin Time and Prothrombin Time Assays

Pooled normal plasma, anticoagulated with 1:9 dilution of 3.8% citrate,was stored at −80° C. until usage. All reagents were pre-warmed to 37°C. and assays were performed in triplicate. For the activated partialthromboplastin time (aPTT) assay 76 μl of plasma was mixed with 76 μlaPTT reagent (aPTT-SA, Helena Laboratories, Beaumont Tex.) and 76 μl oflactadherin diluted into tris-buffered saline. After 10 min. 76 μl of 25mM Ca⁺⁺ was added to start the clotting reaction. Time to fibrin strandformation was measured with a fibrometer. The aPTT reagent was eitherused at full strength or diluted 1:16 in 100 μM ellagic acid, asindicated (to maintain a constant ellagic acid concentration as thephospholipid was diluted).

For the prothrombin time (PT) assay 100 μl plasma was mixed with 100 μllactadherin in tris-buffered saline and 100 μl PT reagent(Thromboplastin-C Plus, Baxter, Miami, Fla.). Because packageinstructions call for use of the PT reagent at a 2:1 ratio with plasma,the PT reagent was supplemented to 16 mM Ca⁺⁺ to achieve themanufacturer's intended final Ca⁺⁺ concentration. The PT reagent wasalso diluted 1:24 in 16 mM Ca⁺⁺, then utilized in the same manner asfull strength PT reagent.

Whole Blood Prothrombin Time Assay

Blood was drawn from healthy, nonsmoking, non-aspirin-using volunteersusing 19-gauge butterfly needles. The first 3-ml of blood was discarded.Subsequently, 20 ml of blood was gently drawn. The blood was rapidlydischarged into a polypropylene tube containing sodium citrate, finalconcentration 10 mM. To suppress contact activation, corn trypsininhibitor was added to the tube (final concentration, 25 μg/ml). Theblood was kept at room temperature, gently mixed by inversionapproximately every 10 min. and was used within 3 hr of collection. Forthe clotting reaction 150 μl aliquots of blood were pre-warmed to 37° C.for 10 min then diluted 1:1 with 10 mM CaCl₂ and 50 pM relipidatedtissue factor (0.38 μM phospholipid). The time to fibrin strandformation was monitored with a fibrometer. Experiments were performed intriplicate.

Results

We hypothesized that the tandem C domains of lactadherin conferphospholipid binding properties that enable it to compete with factorVIII and/or factor V for membrane binding sites and function as ananticoagulant. To test this hypothesis, we performed a competitionmembrane binding experiment in which lactadherin competed withfluorescein-labeled factor VIII for membrane binding sites (FIG. 1A).

For the experiments illustrated in FIG. 1A, fluorescein-labeled factorVIII, 4 nM, was mixed with the indicated concentration of competitor,lactadherin (σ) or annexin V (●) in the presence of 1.5 mM Ca⁺⁺.Lipospheres were added and bound factor VIII was evaluated by flowcytometry after 10 min. Liposphere membrane composition wasPS:PE:PC=4:20:76. The inhibition curve was modeled assuming that theK_(D) of factor VIII with lipospheres was 4.8 nM, the K_(i) forlactadherin 0.5 nM (Reference 16).

The membranes had a composition of 4% PS, 20% PE, with the balance asPC. Bilayers were supported on 2 μm diameter glass microspheres(lipospheres) and binding of factor VIII was evaluated by flow cytometry(Reference 16). Lactadherin effectively competed for all factor VIIIbinding sites with half-maximal displacement occurring at approximately1.5 nM lactadherin. The competition predicted by a mathematical modelapproximated the data when lactadherin was assigned a K_(i) of 0.5 nM.

For comparison with lactadherin, we determined if annexin V wouldcompete for factor VIII binding sites in the same assay (FIG. 1A).Annexin V competed for approximately 20% of factor VIII binding sites ata concentration of 32 nM. These results indicate that lactadherin is amore potent competitor for the phospholipid binding sites of factor VIIIon membranes with 4% PS.

To determine whether lactadherin is also able to recognize thephospholipid binding sites of factor V, we performed similar competitionexperiments with fluorescein-labeled factor V (FIG. 1B).

For the experiments illustrated in FIG. 1B, fluorescein-labeled factorV, 4 nM, was mixed with lactadherin (σ) or annexin V (●) and boundfactor V was evaluated under the conditions described above. Theinhibition curve was modeled assuming that the K_(D) of factor V withlipospheres was 4.3 nM, the K_(i) for lactadherin, 1.0 nM (Reference16).

Lactadherin competed efficiently for the binding sites recognized byfactor V with half-maximal inhibition at an approximately 2-fold higherconcentration. Annexin V competed for only 20% of the factor V bindingsites at concentrations up to 32 nM. The corresponding K_(i) for a curveapproximating the data was 1.0 nM.

We performed factor Xase assays in the presence of increasingconcentrations of lactadherin to determine whether the competition forfactor VIII binding sites would translate into inhibition of the factorXase complex (FIG. 1C).

For the experiments illustrated in FIG. 1C, lactadherin (σ) or annexin V(●) was mixed with factor IXa, 0.1 nM, and factor VI II, 1 nM, andfactor X, 100 nM, prior to the addition of 1 μM sonicated vesicles with1.5 mM Ca⁺⁺ and thrombin. The reaction was stopped after 5 min. andfactor Xa was measured with chromogenic substrate S-2765 in a kineticmicroplate reader. The phospholipid composition of the sonicatedvesicles utilized was the same as for the binding experiments depictedin FIG. 1A. Lactadherin was an effective competitor for binding sites offactor VIII and factor V and potent inhibitor of the factor Xase complexwith half-maximal inhibition at approximately 6 nM and >98% inhibitionat 32 nM. Annexin V was an ineffective inhibitor of the factor Xasecomplex with less than 10% inhibition at 64 nM annexin V. These resultsconfirm that lactadherin is able to inhibit the factor Xase complex,most likely by competing with factor VIIIa and/or factor IXa and factorX for phospholipid binding sites. They do not, however, explain whyannexin V, which also binds to phospholipid membranes with highaffinity, is an ineffective inhibitor under these conditions.

We determined whether lactadherin is capable of inhibiting theprothrombinase complex, in a manner similar to the factor Xase complex(FIGS. 2 A-F, where displayed results are from a single experimentrepresentative of either two or three experiments for all conditions).Prior reports indicate that membrane binding and anticoagulant efficacyof annexin V is related to the PS content of membranes and inverselyrelated to the curvature of phospholipid membranes (Reference 38). Thus,we evaluated vesicles with maximal curvature (sonicated-nominal diameterof 20 nm (Reference 39)) intermediate curvature (extruded-nominaldiameter 73±25 nm (Reference 32)) and minimal curvature (largemultilamellar vesicles-diameters >400 nm (Reference 30)) with both lowPS content (4% PS—FIG. 2A-C) and high PS content (15% PS—FIGS. 2D-F).

For the experiments illustrated in FIGS. 2A-F, sonicated (σ and Δ),extruded (● and μ), and LMV (▪ and □) contained 4% PS (FIGS. 2A-C) or15% PS (FIGS. 2D-F). Effective concentrations of the vesicles forsupporting the prothrombinase complex were identified in phospholipidtitration experiments (FIGS. 2A and 2D). Subsaturating phospholipidconcentrations were selected for inhibition experiments (open symbols inFIGS. 2A and 2D). The indicated phospholipid composition/concentrationwas added to factor Xa, factor Va, prothrombin, and either lactadherinor annexin V. After 5 min. the reaction was quenched with EDTA andthrombin measured with chromogenic substrate S-2238 in a kineticmicroplate reader. Lactadherin was an effective inhibitor of theprothrombinase complex regardless of membrane curvature or PS content(FIGS. 2B and 2E). In contrast, inhibition by annexin V was inverselyrelated to curvature and directly related to PS content (C, F). The Ca⁺⁺concentration was 1.5 mM for sonicated vesicles of 4% PS (cy in A, B, C)and 5 mM for all other conditions.

The effectiveness of the vesicles for supporting prothrombinase complexdecreased as the curvature decreased with sonicated vesicles of 4% PSproducing 90% of the maximum thrombin production at a concentration of0.1 μM phospholipid and much higher concentrations required for extrudedvesicles and large multilamellar vesicles (LMV) (FIG. 2A). We comparedeffects of lactadherin and annexin V on thrombin formation utilizing thephospholipid concentrations (indicated by open symbols in FIG. 2A). Wemaintained the Ca⁺⁺ concentration at 1.5 mM, equivalent to plasma, forsonicated vesicles with 4% PS to test the reproducibility of results inFIGS. 1A-C. However, we increased the Ca⁺⁺ concentration to 5 mM for allother vesicles in order to optimize binding conditions for annexin V.Results showed that >90% inhibition of thrombin production was effectedby lactadherin in sonicated, extruded and LMV. Our results confirmedthat sonicated vesicles provide a substantially more potent surface forthe prothrombinase complex than vesicles of larger diameter,particularly when the PS content is 4%. In contrast, annexin V wasineffective for sonicated vesicles and reached only 25-40% inhibition onextruded vesicles and LMV at 64 nM.

Lactadherin was an efficient inhibitor of the prothrombinase complex onall vesicles containing 15% PS (FIG. 2E). The half-maximal inhibitoryconcentrations of lactadherin varied with vesicle type. The limitingconcentrations of phospholipids for experiments in FIG. 2B facilitatedcomparison of actual inhibition to that predicted by a K_(i) of 1.0 nM,as depicted in FIG. 1B. (The term “K_(i)” is used to indicate onlycompetition of lactadherin for binding sites of factor factor V(a) orfactor VIII(a) when phospholipid binding sites are limiting.) Theinhibition curve approximates the data obtained with sonicated vesicles.The variation in the concentration of lactadherin required for differentvesicle types (FIGS. 2B and 2E) could be rationalized by assuming thatlactadherin binds well to all types of phospholipid vesicles and thatthe higher phospholipid concentrations of extruded vesicles and LMVrequired more lactadherin to saturate the surface. In contrast tolactadherin, annexin V was only an effective inhibitor on LMV when thePS content was 15% (FIGS. 2C and 2F). Under these conditions Annexin Vinhibited approximately 90% of prothrombinase activity at 128 nMconcentration, similar to results in a prior report (References 38 and40). Annexin V was not as effective as lactadherin under any conditionsevaluated. These results indicate that lactadherin efficiently inhibitsthe prothrombinase complex and that, in contrast to Annexin V,inhibition is not closely tied to PS content or to vesicle curvature.

We determined whether inhibition of the factor Xase complex by annexin Vand lactadherin may also be related to PS content of membranes andmembrane curvature (FIGS. 3A-F, where data are from a single set ofexperiments representative of two or three experiments for allconditions).

For the experiments illustrated in FIGS. 3A-F, sonicated (σ and Δ),extruded (● and μ), and LMV (▪ and □) contained 4% PS (FIGS. 3A-C) or15% PS (FIGS. 3D-F). Effective concentrations of the vesicles forsupporting the factor Xase complex were identified in phospholipidtitration experiments (FIGS. 3A and 3D). Subsaturating phospholipidconcentrations were selected for inhibition experiments (open symbols inFIGS. 3A and 3D). Lactadherin was an effective inhibitor of Xaseactivity regardless of membrane curvature or PS content (FIGS. 3B and3E). In contrast, inhibition by annexin V was inversely related tocurvature and directly related to PS content (FIGS. 3C and 3F).Experimental conditions were as those for experiments illustrated inFIGS. 1A-C. The Ca⁺⁺ concentration was 1.5 mM for sonicated vesicles of4% PS (a in FIGS. 3A-C) and 5 mM for all other conditions.

Our results indicate that, like the prothrombinase complex, the factorXase complex is supported at lower concentrations of sonicated vesiclesthan extruded vesicles or LMV(FIGS. 3A and 3D). Also, the inhibition ofthe Xase activity on sonicated vesicles was approximated by a model inwhich lactadherin is assigned a K_(i) of 0.5 nM (FIG. 3B), similar tothe value correlating to direct competition for factor VIII bindingsites (FIG. 1A). Lactadherin inhibited factor Xase complex more than 95%on all vesicle types, similar to inhibition of the prothrombinasecomplex (FIGS. 3B and 3E). However, the difference between thelactadherin concentrations required for inhibition of the factor Xasecomplex on sonicated vs. extruded vesicles and LMV was not as large asfor the prothrombinase complex (FIGS. 2B and 2E). Annexin V was a moreeffective inhibitor of the factor Xase complex than the prothrombinasecomplex with inhibition reaching 80% for LMV of 4% PS (FIG. 3C) and 95%for LMV of 15% PS (FIG. 3F). However, Annexin V remained a poorinhibitor of the factor Xase complex on sonicated vesicles, with <20%inhibition for 4% PS and <50% for 15% PS. Together these resultsindicate that lactadherin is a potent, near-complete inhibitor of theprothrombinase and factor Xase complexes on synthetic membranesregardless of membrane curvature and over a wide range of PS content.

FIGS. 4A-B illustrate inhibition of the factor VIIa-tissue factorcomplex by lactadherin or annexin V. Recombinant tissue factor wasreconstituted into vesicles of composition PS:PE:PC, 4:20:76 bydetergent dialysis with tissue factor:PL ratio of 1:7500. The indicatedconcentrations of phospholipid, with included tissue factor, wereincubated with the indicated concentrations of lactadherin or annexin V,100 pM factor VIIa, 100 nM factor X, and 1.5 mM Ca⁺⁺ for 5 min prior toaddition of EDTA and the chromogenic substrate S-2765. Lactadherininhibited function of the factor VIIa-tissue factor complex efficiently(FIG. 4A) while annexin V was less effective (FIG. 4B). Displayed datawith tissue factor concentration of 1 nM (O) are representative of threeexperiments. Data from experiments with a tissue factor concentration of0.2 nM (μ) and 0.04 nM (σ) are representative of two experiments.

We determined whether Lactadherin might also have the capacity toinhibit the factor VIIa-tissue factor complex (FIG. 4A). Recombinanttissue factor was reconstituted into vesicles containing 4% PS, 20% PEby dialyzing octylthioglucoside away from the tissue factor-phospholipidmixture. Vesicles prepared in this way have curvature comparable toextruded vesicles (Reference 41). Lactadherin inhibited the factorVIIa-tissue factor complex more than 90%. The quantity of lactadherinrequired for 50% inhibition varied with the tissue factor concentration,and corresponding phospholipid concentration. Annexin V was a weakerinhibitor of the factor Vila-tissue factor complex (FIG. 4B) with lessthan 50% inhibition at an annexin V concentration of 64 nM. Theseresults indicate that lactadherin has the capacity to compete formembrane binding sites of blood coagulation proteins other than factor Vand factor VIII.

To directly probe the possibility that lactadherin can compete withvitamin K-dependent blood coagulation proteins for membrane binding, wedetermined whether lactadherin is able to compete withfluorescein-labeled factor IXa (FIG. 5).

For the experiments illustrated in FIG. 5, the active site histidine offactor IXa was derivatized with fluorescein-Glu-Gly-Arg chloromethylketone as described under methods. Four nM fluorescein-labeled factorIXa was mixed with various concentrations of lactadherin or unlabeledfactor IXa in the presence of 5 mM Ca⁺⁺ prior to the addition oflipospheres and evaluation of bound fluorescein-labeled factor IXa asdescribed for FIGS. 1A-C. Lactadherin and unlabeled factor IXa bothcompeted with labeled factor IXa for membrane binding sites. Results aredisplayed with the abscissa on a log scale to enable direct comparison.Displayed data are representative of two experiments.

Fluorescein-labeled factor IXa bound to lipospheres with a dissociationconstant of approx. 0.5 μM (data not shown), consistent with priorreports of the membrane-binding affinity of factor IX/factor IXa.Unlabeled factor IXa competed with fluorescein-labeled factor IXaindicating that membrane binding was not enhanced by derivatization byfluorescein-Glu-Gly-Arg chloromethyl ketone at the active site.Lactadherin competed with fluorescein-labeled factor IXa for membranebinding sites to the same extent as unlabeled factor IXa. However,half-maximal inhibition occurred at approximately 4 nM lactadherin vs.300 nM factor IXa.

In order to confirm that the mechanism through which lactadherininhibits membrane-bound blood coagulation complexes is via competitionfor membrane binding sites, we performed experiments with varyingphospholipid concentrations (FIGS. 6A-C).

For the experiments illustrated in FIGS. 6A-C, factor Xase complex (FIG.6A) and prothrombinase complex (FIG. 6B) function on sonicated vesicleswas evaluated at 0.1 (●), 0.5 (□), and 2.5 (σ) μM phospholipidconcentrations in the presence of 1.5 mM CaCl₂. Function of the factorXase complex in the absence of phospholipid was also evaluated (FIG.6C). Inhibition by lactadherin at various concentrations was measuredusing two step amidolytic assays for the production of factor Xa (FIGS.6A and 6C) or thrombin (FIG. 6B) as described above. The inhibitoryconcentration of lactadherin was directly related to phospholipidconcentration. In the absence of phospholipid (FIG. 6C) lactadherin wasan inefficient inhibitor of the factor Xase complex. Data obtained atvarious phospholipid concentrations were normalized for comparison ofrelative inhibition by lactadherin. Phospholipid composition wasPS:PE:PC 4:20:76. Displayed data are from a single set of experiments(FIGS. 6A and 6B) or a single experiment representative of threeexperiments (FIG. 6C).

When the phospholipid vesicle concentration was limiting, lactadherininhibited 50% activity of the factor Xase complex (FIG. 6A) and theprothrombinase complex (FIG. 6B) at a concentration of approx. 2 nM. Theconcentration required increased for each increment in the phospholipidconcentration indicating that the required lactadherin is related to thephospholipid concentration rather than the concentrations of bloodcoagulation proteins. Furthermore, when the phospholipid concentrationwas 2.5 μM the factor Xase complex and prothrombinase complex maintainedmore than 90% activity at 8 nM lactadherin, a concentration thatinhibits activity more than 90% when the phospholipid concentration islimiting.

As noted above, we also evaluated the effect of lactadherin on thefactor Villa-factor IXa complex in the absence of phospholipid (FIG.6C). The concentrations of factor Villa, factor IXa, and factor X werechosen to be at or below their apparent K_(D)'s or K_(M), respectively,thus optimizing the sensitivity of the reaction to inhibitory action oflactadherin. Lactadherin caused less than 50% inhibition atconcentrations up to 512 nM. Thus, lactadherin is at least 1,000-timesbetter inhibitor in the presence of phospholipid membranes.

These results indicate that the only mechanism through which lactadherininhibits the factor Xase and prothrombinase complexes at theconcentrations employed is by competitive occupation of phospholipidbinding sites and that protein-protein complexes between lactadherin andfactor VIIIa, factor Va, factor IXa, or factor X do not occur, or do notsignificantly inhibit function of these enzyme complexes.

To further investigate the dependence of lactadherin's inhibitoryproperties on phospholipid concentration, we evaluated inhibition ofplasma clotting in activated partial thromboplastin time (aPTT) andprothrombin time (PT) assays. Commercial aPTT and PT reagents containhigh concentrations of phospholipids of unknown or unspecifiedcomposition. Lactadherin inhibited the aPTT assay by approx. 10% at aconcentration of 1,000 nM and had no effect at 100 nM or lowerconcentrations. However, when the aPTT reagent was diluted to 6% of theoriginal concentration the aPTT was prolonged 5%, 20%, and 1000% atconcentrations of 10, 100, and 1,000 nM, respectively. Similarly, 1,000nM lactadherin inhibited the prothrombin time by <20% when the PTreagent was present at 50% of manufacturer's suggested usage. However,when the PT reagent was diluted to 2% of the stock concentration the PTwas prolonged 5%, 30%, and 500% by lactadherin concentrations of 10,100, and 1,000 nM, respectively. These results are consistent with themodel in which the major mechanism through which lactadherin inhibitsblood coagulation enzyme complexes is through competition forphospholipid binding sites. Further studies, with defined phospholipidmembranes are recommended to determine whether the apparent K_(i)'s forinhibition of the isolated prothrombinase and Xase complexes correlateto inhibitory concentrations for plasma.

The results above, showing inhibition of the factor Xase, theprothrombinase and the factor Vila-tissue factor complexes support thehypothesis that lactadherin would inhibit the rate at which whole bloodclots. To test this prediction, we utilized a modified whole bloodprothrombin time (FIG. 7).

For the experiments illustrated in FIG. 7, fresh whole blood wasanticoagulated with 10 mM citrate and 25 μg/ml corn trypsin inhibitor(to minimize activation of the intrinsic pathway prior to theprothrombin time assay) in a polypropylene tube. After addition ofindicated quantities of lactadherin (σ) or annexin V (●), the blood wasdiluted 1:1 with 50 pM tissue factor and 10 mM Ca⁺⁺. Time to fibrinstrand formation was measured with a fibrometer. Blood coagulation wasinitiated by simultaneous addition of calcium and 50 pM tissue factorprepared as in FIGS. 4A-B. Results are displayed as the ratio ofprothrombin time for blood treated with annexin V or lactadherin toprothrombin time for control blood from the same donor, with the sameelapsed time since phlebotomy. Displayed results are mean±SD fortriplicate samples from a single experiment representative of threeexperiments.

In the absence of lactadherin or annexin V, the time to clot variedbetween 1200 and 2000 seconds for different donors. Lactadherin andannexin V led to prolongation of the clotting time and the prolongationwas similar over a concentration range of 0-20 nM. However, atconcentrations of 40 nM and above lactadherin led to progressivelylonger inhibition of blood clotting. The clotting time was prolongedapproximately 3-fold at 100 nM lactadherin, but only about 1.5-fold byannexin V. These results indicate that Lactadherin competes for bindingsites on cell membranes to inhibit blood coagulation in a manner similarto inhibition of isolated blood coagulation complexes on phospholipidvesicles.

Our results indicate that lactadherin binds to PS-containing membraneswith sufficient affinity to compete with blood coagulation proteins. Thephospholipid-binding competition makes lactadherin a potent inhibitor ofthe prothrombinase, the factor Xase, and the factor VIIa-tissue factorcomplexes of blood coagulation. Because the quantity of lactadherinnecessary to inhibit these enzyme complexes is proportional to thephospholipid concentration employed, and because lactadherin does notefficiently inhibit the phospholipid-free factor VIIIa-factor IXacomplex, the major mechanism of inhibition involves blocking of thephospholipid surface rather than formation of inhibitory protein-proteincomplexes. Inhibition of whole blood prothrombin time indicates thatlactadherin binds to platelet membranes to inhibit blood coagulation ina mechanism similar to inhibition of reconstituted enzyme complexes onphospholipid vesicles.

The enzyme complexes of blood coagulation assemble and functionefficiently only on a membrane surface. PS-containing membranes serve toincrease the apparent affinity of the respective cofactors, factor Villaand factor Va, for the enzymes, factor IXa and factor X, and of thecofactor-enzyme complexes for the substrates, factors X and prothrombin,respectively (Reference 42). The membranes also serve as allostericactivators of the enzyme-cofactor complex (Reference 29). PS-containingmembranes also support anticoagulant activity that modulates theprocoagulant activity. For example, protein C is activated by thethrombin-thrombomodulin complex on PS-containing membranes (Reference43) and activated protein C inactivates factor Va and factor Villa onPS-containing membranes (Reference 44). The membranes of quiescent bloodcells do not display the PS necessary to enable assembly and function ofthe procoagulant enzyme complexes (References 45-46). Rather, it isexposed only after cells are stimulated or undergo apoptosis (Reference47). In the setting of a tissue injury, the procoagulant membranes areprobably on the surface of platelets that have adhered to the damagedtissues (Reference 48). The absence of a PS-containing phospholipidmembrane effectively prevents function of the complexes. Thus, blockingthe PS-containing phospholipid binding sites on platelets appears to bea potential mechanism for preventing blood coagulation or altering theprocoagulant/anticoagulant balance. Additional investigation isrecommended to determine whether lactadherin has this physiologicfunction.

Several proteins have been identified that can influence bloodcoagulation via interaction with phospholipid membranes. The hypothesisthat lactadherin might function in this manner was based on the homologybetween the discoidin-type domains of lactadherin and those of factorsVIII and V, together with the previously defined membrane-bindingproperties of lactadherin (Reference 6). Annexin V, the most thoroughlystudied of these proteins, binds to PS-containing membranes with highaffinity (Reference 25). However, annexin V binds poorly to curvedmembranes (Reference 40) requires supraphysiologic Ca⁺⁺ concentrationsfor optimal binding, and inhibits less than 80% of procoagulant functionon endothelial cell membranes unless the concentration exceeds 200 nM(Reference 49). Likewise, annexin V is an incomplete inhibitor of thefactor Xase complex on platelet membranes (Reference 50). Our results,indicating inhibition of the prothrombinase complex exceeds 80% at 60 nMannexin V only when the curvature of the membrane is minimal and the PScontent is 15%, are in agreement with these prior studies.β₂-glycoprotein I binds to PS-containing membranes and other negativelycharged lipid-containing particles such as lipoproteins. Purifiedβ₂-glycoprotein I partially inhibits prothrombinase activity on purifiedplatelets or phospholipid vesicles (Reference 51). However,β₂-glycoprotein I may be a more efficient inhibitor of the anticoagulantreaction in which activated protein C cleaves factor V or factor Va on aphospholipid membrane (Reference 52). β₂-glycoprotein I bound tophospholipid is the primary antigen of lupus-type anticoagulants(Reference 53). When an antibody links two β2-glycoprotein I moleculesthe membrane-binding affinity is increased and β₂-glycoprotein becomes amore potent in vitro anticoagulant (Reference 54). Whetherβ₂-glycoprotein I has a physiologic function influencing procoagulant oranticoagulant membrane interactions remains unknown (Reference 55). Thephysiologic relationship of these proteins and lactadherin to bloodcoagulation is a likely field for further investigation.

Factor VIII binds to sites on phospholipid membranes with remarkablespecificity. The specificity is best illustrated by the failure of otherlipid binding proteins to compete with factor VIII for these sites(Reference 16). Even factor V, with structural homology and equivalentaffinity for phospholipid membranes competes for only a fraction of thesites recognized by factor VIII. The capacity of lactadherin to competefor membrane binding sites of factor VIII, also to inhibit both thefactor Xase complex and the prothrombinase complex indicates thatlactadherin is more promiscuous than factor VIII with regard tophospholipid binding sites (Reference 16). Inhibition of the factorVIIa-tissue factor complex indicates that lactadherin has the capacityto compete for the vitamin K dependent proteins, factor VIla and/orfactor X. The contrast between lactadherin and annexin V with regard tocompeting for membrane sites of both high and low PS content and varyingmembrane curvature indicate that lactadherin is also more promiscuous inits membrane requirements than annexin V. To facilitate understanding ofthese properties we have initiated studies to characterize the membranebinding properties of lactadherin vs. those of factor VIII and factor V.The results indicate that lactadherin resembles factor VIII and factor Vin specific binding to PS and curvature-dependent membrane binding.However, lactadherin differs in having a lower PS requirement and noapparent requirement for PE on membranes with low PS content.

Comparing the displacement of factor VIII and factor V by lactadherinwith the competition predicted from the simplest mathematical model(FIGS. 1A-C and 3A-F) supports two conclusions. First, lactadherin is abetter competitor for factor VIII binding sites than for factor Vbinding sites with a K_(i) that is two-fold lower. More potentcompetition for factor VIII binding sites correlated with a two-foldlower K_(i) for the factor Xase complex vs. the prothrombinase complexnot only under conditions where phospholipid was limiting but also whenphospholipid was not limiting (FIG. 6). The lower K_(i) value suggeststhat lactadherin binds with higher affinity to phospholipid bindingsites of factor VIII vs. those of factor V. The second conclusion isthat the experimental data did not precisely conform to the curvespredicted by the mathematical model. The assumption of the model wasthat lactadherin competed for a single class of phospholipid bindingsites with factor VIII or factor V. In other studies, we have furthercharacterized the interaction of lactadherin with phospholipid bindingsites. Our results indicate that lactadherin recognizes at least2-classes of phospholipid binding sites so that both association anddissociation are kinetically complex events. The ability to interactwith multiple classes of phospholipid binding sites explains part of thevariation from the simple model as well as the mechanism underlying thecapacity to compete with factor VIII and factor V with differentapparent K_(u)'s.

The concentrations of lactadherin required to inhibit the whole bloodprothrombin time were somewhat higher than the concentrations necessaryto inhibit isolated enzyme complexes (compare FIGS. 2-4 vs. FIG. 6). Ourdata do not indicate whether these apparent discrepancies inconcentration reflect lactadherin binding to a plasma protein thatpartially competes with binding sites on the membranes of platelets orother cells, or whether lactadherin may have a lower affinity for cellmembranes vs. phospholipid vesicles.

The results of our experiments outlined above indicate that lactadherincould serve as a physiologic or pharmacologic anticoagulant. In newborncalves, the plasma concentration rises from 0.07 μg/ml before feeding to1.2 μg/ml after feeding, suggesting that intact lactadherin is absorbedacross the GI tract and that sufficient lactadherin circulates in theblood under these conditions to have a measurable in vitro anticoagulanteffect (Reference 7). Lower concentrations of lactadherin have beenmeasured in the serum of women with metastatic breast carcinoma but notin the serum of normal controls (Reference 56). The plasma levels inpregnant or lactating mammals have not been reported. However, it isplausible that sufficient lactadherin may be secreted into the blood ormay “leak” from mammary glands to provide an anticoagulant effect duringpregnancy or lactation. The relatively small size of lactadherinsuggests that it freely traverses the placental barrier and could affectthe procoagulant/anticoagulant balance of a developing fetus. Thepresence of lactadherin on the apical surfaces of secretory epitheliaother than breast tissue suggests that lactadherin may circulate inblood even during the non-pregnant state. Thus, it is plausible thatlactadherin could provide a physiologic anticoagulant function under avariety of circumstances. Our data support the conclusion thatlactadherin is a candidate for development as a pharmacologicanticoagulant. Lactadherin functions at steps that are early in thecoagulation pathway and is apparently more potent than annexin V, theonly other tested agent that functions by a similar mechanism.

In view of the above, we believe that:

-   -   a. Lactadherin may be used as an anticoagulant in human beings.        Because of its unique mechanism of action it may prove useful in        specific clinical situations such as diffuse intravascular        coagulation of obstetrics or septic shock. The natural method in        which it is produced by mammary glands and secreted in milk        suggests that it would prove much cheaper to produce than most        protein drugs. Depending upon its efficacy, it might have a        market as broad as all pregnant women with blood clots or        patients with atherosclerosis who are experiencing unstable        angina or a heart attack.    -   b. Lactadherin may be used as an anti-inflammatory agent in        human beings, particularly in circumstances where blood        coagulation is associated with inflammatory reactions including        cell signaling mediated by coagulation enzymes and tissue        factor. An example of a clinical situation where lactadherin may        have anti-inflammatory properties is in sepsis.    -   c. Lactadherin may be used to measure procoagulant phospholipid        that circulates in the blood of patients. Because lactadherin        completely blocks access to procoagulant phospholipid, it may be        applied to laboratory assays as a method to block access of        blood coagulation proteins from all circulating procoagulant        phospholipids and to determine the residual coagulation        activity.    -   d. Lactadherin may be used as a laboratory reagent to remove        circulating phospholipids from the plasma of patients in order        to measure the coagulation potential of plasma in the presence        of defined phospholipid. For example, lactadherin might be        covalently linked to a matrix such as sepharose and procoagulant        lipids removed by passage over the matrix. It might then be        possible to provide a sensitive assay for lupus-type        anticoagulants.    -   e. Measurement of blood lactadherin concentrations may be useful        to guide anti-coagulant or anti-inflammatory therapy. For        example, lactadherin may be a natural anticoagulant during        pregnancy, lactation, or neonatal life. Measurement may predict        risk of thrombosis or guide therapy in case of thrombosis.

A pharmaceutical composition including lactadherin or a fragment oflactadherin, or a functionally equivalent agent thereof, may beprepared, in a conventional manner. In particular, a pharmaceuticalcomposition made in accordance with the present invention would includelactadherin or a fragment of lactadherin, or a functionally equivalentagent thereof, in an amount sufficient to provide therapeutic and/orprophylactic benefit, in combination with one or more pharmaceuticallyor physiologically acceptable carriers, diluents or excipients.Compositions of the present invention may be formulated for anyappropriate manner for administration, including, for example, oral,nasal, intravenous or intramuscular administration. Appropriate dosages,duration and frequency of administration would be determined by knownfactors, such as the condition of the patient, the type and severity ofthe disease and the method of administration.

While this invention has been described as having preferred ranges,steps, materials, or designs, it is understood that it is capable offurther modifications, uses and/or adaptations of the inventionfollowing in general the principle of the invention, and including suchdepartures from the present disclosure as those come within the known orcustomary practice in the art to which the invention pertains, and asmay be applied to the central features hereinbefore set forth, and fallwithin the scope of the invention and of the limits of the appendedclaims.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of blocking or reducing access to a procoagulant molecule bya coagulation molecule, comprising; subjecting a procoagulant moleculeto lactadherin, a fragment of lactadherin, a functional equivalent oflactadherin, or a functional equivalent of a fragment of lactadherin. 2.The method of claim 1, wherein: a cell membrane or a fragment thereofcomprises the procoagulant molecule.
 3. The method of claim 1, wherein:the procoagulant molecule comprises a phospholipid or a lipoprotein. 4.The method of claim 3, wherein: the coagulation molecule comprises ablood coagulation protein.
 5. The method of claim 3, wherein: thecoagulation molecule is selected from the group consisting of factor V,factor Va, factor VIIa, factor VIII, factor VIIIa, faxtor IXa, andfactor X.
 6. The method of claim 4, wherein: the procoagulant moleculecomprises phosphatidylserine (PS).
 7. A method of blocking or reducingbinding of a ligand to a cell membrane binding site, comprising:subjecting a cell membrane binding site to lactadherin, a fragment oflactadherin, a functional equivalent of lactadherin, or a functionalequivalent of a fragment of lactadherin.
 8. The method of claim 7,wherein: the ligand comprises a blood coagulation molecule selected fromthe group consisting of factor V, factor Va, factor VIIa, factor VIII,factor Villa, faxtor IXa, and factor X.
 9. The method of claim 8,wherein: the cell membrane site comprises a phospholipid.
 10. A methodof inhibiting or slowing blood coagulation, comprising: subjecting apredetermined amount of blood to an effective amount of lactadherin, afragment of lactadherin, a functional equivalent of lactadherin, or afunctional equivalent of a fragment of lactadherin.
 11. A method ofinhibiting or slowing blood clotting, comprising: administering to asubject in need thereof an effective amount of lactadherin, a fragmentof lactadherin, a functional equivalent of lactadherin, or a functionalequivalent of a fragment of lactadherin.
 12. A method of preventing orreducing inflammation, comprising: administering to a subject in needthereof an effective amount of lactadherin, a fragment of lactadherin, afunctional equivalent of lactadherin, or a functional equivalent of afragment of lactadherin.
 13. A method of inhibiting or slowing bloodcoagulation, comprising: administering to a subject in need thereof aneffective amount of lactadherin, a fragment of lactadherin, a functionalequivalent of lactadherin, or a functional equivalent of a fragment oflactadherin.
 14. An anticoagulant reagent, comprising: lactadherin, afragment of lactadherin, a functional equivalent of lactadherin, or afunctional equivalent of a fragment of lactadherin.
 15. A pharmaceuticalcomposition, comprising the reagent of claim 14 and a pharmaceuticallyacceptable carrier or diluent.
 16. A method of removing a phospholipidfrom a biological fluid, comprising: a) providing a biological fluidincluding, or suspect of including, a phospholipid; b) subjecting thebiological fluid to a suitable amount of a binding agent selected fromthe group consisting of lactadherin, a fragment of lactadherin, afunctional equivalent of lactadherin, and a functional equivalent of afragment of lactadherin; c) allowing binding between the phospholipidand the binding agent; and d) removing the binding agent with anyphospholipid bound thereto.
 17. The method of claim 16, wherein: thebinding agent is provided in a sepharose matrix.
 18. A kit for removinga phospholipid from a biological fluid, comprising: a) a binding agentselected from the group consisting of lactadherin, a fragment oflactadherin, a functional equivalent of lactadherin, and a functionalequivalent of a fragment of lactadherin; and b) instructions for use ofthe binding agent.
 19. The kit of claim 18, further comprising: asepharose matrix comprising the binding agent.